U.S. patent number 7,547,384 [Application Number 10/508,482] was granted by the patent office on 2009-06-16 for electrochemical detector systems.
This patent grant is currently assigned to Millipore Corporation. Invention is credited to Elizabeth Ann Keenan.
United States Patent |
7,547,384 |
Keenan |
June 16, 2009 |
Electrochemical detector systems
Abstract
The present invention concerns methods for detecting
micro-organisms in a sample, and apparatus comprising hollow fibre
filter membranes for same.
Inventors: |
Keenan; Elizabeth Ann (Bolton,
GB) |
Assignee: |
Millipore Corporation
(Billerica, MA)
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Family
ID: |
9934262 |
Appl.
No.: |
10/508,482 |
Filed: |
April 2, 2003 |
PCT
Filed: |
April 02, 2003 |
PCT No.: |
PCT/GB03/01445 |
371(c)(1),(2),(4) Date: |
February 17, 2005 |
PCT
Pub. No.: |
WO03/087398 |
PCT
Pub. Date: |
October 23, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050126908 A1 |
Jun 16, 2005 |
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Foreign Application Priority Data
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Apr 4, 2002 [GB] |
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0207813.7 |
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Current U.S.
Class: |
205/778;
204/403.01 |
Current CPC
Class: |
C12Q
1/24 (20130101); B01D 61/22 (20130101); B01D
63/02 (20130101); B01D 2311/24 (20130101) |
Current International
Class: |
G01N
27/327 (20060101) |
Field of
Search: |
;205/778 ;204/403.01
;422/44-48 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2 809 969 |
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FR |
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2135902 |
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GB |
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2 352 652 |
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Feb 2001 |
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GB |
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87/03690 |
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Jun 1987 |
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WO |
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94/25848 |
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Nov 1994 |
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WO |
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96/04067 |
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Feb 1996 |
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WO |
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98/04675 |
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Feb 1998 |
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WO |
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00/23792 |
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Apr 2000 |
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WO |
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02/062941 |
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Aug 2002 |
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WO |
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Other References
Proc. SPIE vol. 4265; pp. 65-74; Dalibor Hodko et al.; "Detection
of Pathogens Using On-Chip Electrochemical Analysis of PCR
Amplified DNA Molecules". cited by other .
European communication dated Dec. 21, 2006. cited by other .
European communication dated Sep. 5, 2007. cited by other .
European communication dated Mar. 5, 2008. cited by other .
Office actions dated Apr. 5, 2006, Jul. 12, 2006, Mar. 16, 2007,
Nov. 5, 2007, Jul. 25, 2007, May 1, 2008 (corresponding U.S. Appl.
No. 10/467,440). cited by other .
Office actions dated Apr. 24, 2008 (corresponding U.S. Appl. No.
11/382,725). cited by other.
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Primary Examiner: Noguerola; Alex
Attorney, Agent or Firm: Nields, Lemack & Frame, LLC
Claims
The invention claimed is:
1. A method for detecting a micro-organism in a sample, comprising
the steps of: i) passing said sample through the sample inlet of a
filter device comprising a plurality of hollow fibre filter
membranes and at least one detector system, said membranes having
first and second ends, an outer surface and an inner surface
defining a lumen, said first end of each of said membranes being
open and communicating with said sample inlet and flow through said
second end of each of said membranes being restricted such that
said sample mixture is filtered through said membranes, leaving a
filtrand in said lumen of said membranes, said at least one
detector system comprising an electrode which runs through said
lumen of said membranes; ii) optionally resuspending said filtrand
from said lumen of said membranes; iii) detecting with said at
least one detector system the presence of micro-organisms in said
filtrand; and iv) correlating the results of detection step (iii)
with the presence of said micro-organism in said sample.
2. A method according to claim 1, step (i), additionally comprising
the step of detecting with said at least one detector system the
presence of micro-organisms in said sample.
3. A method according to claim 2, correlation step (iv) comprising
correlating the results of detection step (i) and detection step
(iii) with the presence of said micro-organism in said sample.
4. A method according to claim 1, 2 or 3, wherein said at least one
detector system comprises a plurality of electrodes.
5. A method according to claim 4, wherein the surface of at least
one of said electrodes of a first detector system has been modified
such that a plurality of first members of a specific binding pair
depends therefrom.
6. A method according to claim 4, wherein said filter device
comprises first and second detector systems, wherein the surface of
at least one of said electrodes of a first detector system has been
modified such that a plurality of first members of a specific
binding pair depends therefrom, and the surface of at least one of
said electrodes of a second detector system has been modified such
that a plurality of second members of a specific binding pair
depends therefrom.
7. A method according to claim 4, wherein the surface of at least
one of said electrodes of at least one additional detector system
has been modified such that a plurality of first members of at
least one additional second specific binding pair depends
therefrom.
8. A method according to any one of claims 1-3, wherein said at
least one detector system detects at least one of the group
consisting: bio-luminescence, electro-chemical activity,
fluorescence, chemi-luminescence, radiochemicals, radioisotopes,
alpha particles, beta particles, gamma rays, antibody-antigen
interactions, protein-protein interactions, protein-enzyme
interactions, nucleic acid hybridisation.
9. A method according to claim 1, wherein said at least one
detector system comprises at least one gold electrode.
10. A method according to claim 1, wherein said electro-chemical
detector system comprises at least one long chain polymer
electrode.
11. A method according to claim 1, wherein said at least one
detector system is an electrochemical biosensor.
12. A filter device comprising a plurality of hollow fibre filter
membranes and at least one detector system, said membranes having
first and second ends, an outer surface and an inner surface
defining a lumen, said first end of each of said membranes being
open and communicating with said sample inlet and flow through said
second end of each of said membranes being restricted such that
said sample mixture is filtered through said membranes, leaving a
filtrand in said lumen of said membranes, said at least one
detection system comprising an electrode which runs through said
lumen of said membranes.
13. The filter device of claim 12, wherein said at least one
detector system comprises a plurality of electrodes.
14. The filter device of claim 12, wherein said at least one
detector system comprises at least one gold electrode.
15. The filter device of claim 12, wherein said at least one
detector system comprises at least one long chain polymer
electrode.
16. The filter device of claim 12, wherein said at least one
detector system comprises an electrochemical biosensor.
17. The filter device of claim 12, wherein the surface of at least
one of said electrodes of a first detector system has been modified
such that a plurality of first members of a specific binding pair
depends therefrom.
18. The filter device of claim 12, wherein said filter device
comprises first and second detector systems, wherein the surface of
at least one of said electrodes of a first detector system has been
modified such that a plurality of first members of a specific
binding pair depends therefrom, and the surface of at least one of
said electrodes of a second detector system has been modified such
that a plurality of second members of a specific binding pair
depends therefrom.
19. The filter device of claim 12, wherein the surface of at least
one of said electrodes of at least one additional detector system
has been modified such that a plurality of first members of at
least one additional second specific binding pair depends
therefrom.
20. The filter device of claim 12, wherein said at least one
detector system detects at least one of the group consisting:
bioluminescence, electrochemical activity, fluorescence,
chemi-luminescence, radiochemicals, radioisotopes, alpha particles,
beta particles, gamma rays, antibody-antigen interactions,
protein-protein interactions, protein-enzyme interactions, nucleic
acid hybridization.
Description
As rigid product quality regulations become enforced within the
food, water and beverage industry, it is becoming increasingly
necessary to improve on the methods used to detect contaminants
within product processes, since current methods are labour
intensive and require long time periods to generate results. The
ability to filter greater volumes using hollow fibre membrane
technology (such as in WO 01/11006) provides a method of
concentrating the number of contaminants from a much greater and
more representative sample volume and increases the level of
sensitivity achieved. The limitation to this technology is the
method used to identify contaminants.
The requirement for the identification of a single cell within a
volume of liquid eg. beer, water or fruit juice is limited by the
current techniques applied. To increase the sensitivity of
detection for microbial contamination in a sample, methods using
biosensors, PCR (polymerase chain reaction), or immunological
technology have been developed. However, these technologies all
require the analysis and detection of contaminants from a small
volume of the sample liquid (typically in ml-.mu.l range), which is
usually unrepresentative of the large volume of liquid being
sampled (eg from a 20 000 L fermenter), often resulting in a
failure to detect contaminants in the sample.
The potential combination of the existing membrane technology seen
in WO 01/11006 with newly developing nanoscale based systems would
lead to increased sample handling with real time analysis
capability. Typical nanoscale systems can utilise electrochemical
technology, biosensor technology, nanowires, self-assembled
monolayers (SAMs), and high sensitivity miniaturised detector
systems. The incorporation of single or multiple fibre array
detector systems combining wire electrochemistry or membrane
surface coating technology and biosensor applications onto hollow
fibre membrane would greatly facilitate the analysis and detection
of contaminants for any given sample. The ability to accommodate
reverse flow generation within the membrane filtration system
enables biomolecules that have been undetected during the first
filtration phase to be re-exposed to the detector surface, thereby
increasing the detector sensitivity.
According to the present invention, there is a provided a method
for detecting a micro-organism in a sample, comprising the steps
of: i) passing said sample through the sample inlet of a filter
device comprising a plurality of hollow fibre filter membranes and
at least one detector system, said membranes having first and
second ends, an outer surface and an inner surface defining a
lumen, said first end of each of said membranes being open and
communicating with said sample inlet and flow through said second
end of each of said membranes being restricted such that said
sample mixture is filtered through said membranes, leaving a
filtrand in said lumen of said membranes, said at least one
detector system being at least partially contained within said
lumen of said membranes; ii) optionally resuspending said filtrand
from said lumen of said membranes; iii) detecting with said at
least one detector system the presence of micro-organisms in said
filtrand; and iv) correlating the results of detection step (iii)
with the presence of said micro-organism in said sample.
The method may comprise the additional step of detecting with at
least one detector system the presence of micro-organisms in the
sample mixture, before or during the filtration step.
The hollow fibre membranes may be composed of a variety of polymers
such as polypropylene, polyethersulphone and cellulose acetate.
The detector system may be an electrochemical detector system and
may comprise a plurality of electrodes. Examples of suitable
electrodes include gold electrodes, and long chain polymer
electrodes.
The detector system may be an electrochemical biosensor.
The surface of at least one of the electrodes of the detector
system may be modified such that a plurality of first members of a
specific binding pair depends therefrom.
A "Member of a Specific Binding Pair" is one of two different
molecules, having an area on the surface or in a cavity which
specifically binds to and is thereby defined as complementary with
a particular spatial and polar organization of the other molecule.
The members of the specific binding pair (sbp) are referred to as
ligand and receptor (antiligand), sbp member and sbp partner, sbp
members or the like. These are usually members of an immunological
pair such as antigen-antibody, although the term does have a
broader meaning encompassing other specific binding pairs, such as
biotin-avidin, hormones-hormone receptors, nucleic acid hybrids
(e.g. DNA-DNA, DNA-RNA, RNA-RNA), IgG-protein A.
The modification of the surface chemistry of either the gold or
polymer electrode facilitates the attachment of biomolecules such
as antibodies, enzymes and nucleic acids. These biomolecules may
specifically interact with second members of the specific binding
pair within the fluid stream (e.g. an antigen if the first member
of the specific binding pair is an antibody), generating an
electrically active complex which can be detected, amplified,
quantitated and displayed using an amplifier, potentiostat, and
computer (running appropriate data acquisition software)
set-up.
The surface of at least one of the electrodes of a second detector
system may be modified such that a plurality of first members of a
second specific binding pair depends therefrom.
The surface of at least one of the electrodes of at least one
additional detector system may be modified such that a plurality of
first members of at least one additional specific binding pair
depends therefrom. Since the use of additional specific binding
pairs facilitates the specific detection of additional
micro-organisms within the sample, the use of multiple electrodes
(each coated with a different specific binding pair) within the
filter device, would facilitate the specific detection of multiple
micro-organisms within a sample.
The detector system may detect at least one of the group
comprising: bioluminescence, electrochemical activity,
fluorescence, chemi-luminescence, radiochemicals, radioisotopes,
alpha particles, beta particles, gamma rays, antibody-antigen
interactions, protein-protein interactions, protein-enzyme
interactions, protein-nucleic acid hybrids, nucleic acid
hybrids.
The filter device may comprise a plurality of hollow fibre filter
membranes and at least one detector system, said membranes having
first and second ends, an outer surface and an inner surface
defining a lumen, said first end of each of said membranes being
open and communicating with said sample inlet and flow through said
second end of each of said membranes being restricted such that
said sample mixture is filtered through said membranes, leaving a
filtrand in said lumen of said membranes, said at least one
detection system being at least partially contained within said
lumen of said membranes.
The invention will be further apparent from the following
description, with reference to the accompanying Figures, which
show, by example only, forms of the filter device and testing
methods.
Of the Figures:
FIG. 1 shows a hollow fibre membrane filter device;
FIG. 2 shows a gold wire electrochemical detector; and
FIG. 3 shows a hollow fibre electrochemical detector set-up.
In a first embodiment of the present invention the electrochemical
detector system (10) may comprise a three electrode set-up where
the electrodes are designated working (20), reference (30) and
counter (40). The working electrode (20) is gold and runs through
the lumen (50) of the hollow fibre membrane (60) (FIG. 2), the
counter electrode (40) is placed partially into the lumen (50) of
the membrane (ca 10 mm), and the reference electrode (30) is
connected externally to the reference material (70) (test sample).
The working and counter electrodes (20, 40) are then connected to a
gold contact pad (80) which is located on the outer rim (90) of the
hollow fibre housing (100) used to support the hollow fibre
membranes (60) (FIG. 1). The contact pads (80) for both the working
and counter electrodes (20, 40) are applied to the end of the
hollow fibre housing (100) by screen printing during injection
moulding of the filter housing. Other available procedures include
standard photolithographic lift off procedures, and the use of
silver conducting paint. After screen printing into the injection
moulding of the end cap tool, the electrodes (20,40) are then
connected to the contact pads (80) using silver conducting paint
followed by connection of standard timed copper wires (130, 140)
being connected to the electrode contact pads (80). The copper
wires (130, 140) are connected at one end to a Molex crimp (150)
which is slotted into a Molex Shell/PCB header (170). A direct
connection is made from the Molex Shell/PCB header (170) to a
pre-amp (180) using standard tinned copper wire (190). The pre-amp
(180) is connected to a potentiostat (200) via an amplifier (210).
A computer (215) is connected to the potentiostat (200).
Potentiostats establish a specific potential across a
metal-electrolyte interface, usually so that the current passed
through the interface can be measured. Many potentiostats have the
capability of operating as a galvanostat. In this form of operation
a predetermined constant current may be forced through the metal
electrolyte interface and the potential across it measured. The
potentiostat can convert the signal into measurable data which is
processed on a computer (215) running LabVIEW data acquisition
software. The reference electrode (30) can be pseudo-floating, i.e.
it can be connected externally to the pre-amp (180) and is not part
of the filter device (10) (FIG. 3).
The use of an electrochemical detector using a bare gold wire
allows for the detection (using linear sweep, cyclic voltammetry or
chronoamperometry) of the most simple of electrically active
analytes (e.g. protons for pH measurement).
In a second embodiment of the present invention, the sensitivity
and selectivity of the detector system can be increased by
converting the device into an electrochemical biosensor. A
biosensor is generally a metal or polymer substrate with a
modification to its surface chemistry, which allows for the
immobilisation of a first member of specific binding pair e.g.
biomolecules such as antibodies, enzymes and nucleic acids. These
biomolecules then interact with second members of the specific
binding pair within the fluid stream, generating an electrically
active complex which can be detected electrochemically. In order to
bind the first member of a specific binding pair, the surface of
the gold electrode is modified by silinization, either through
immersion into a silane solution, or alternatively by vapour
deposition. Alternatively, self-assembled monolayers (SAMs) can be
prepared using different types of molecules and different
substrates--examples include alkylsiloxane monolayers, fatty acids
on oxidic materials, and alkanethiolate monolayers. Alkanethiolate
monolayers are especially useful since they adsorb with high
efficiency to gold electrodes. Alkanethiolate is a molecule which
is essentially an alkane chain, typically with 10-20 methyl units,
and a head group with a strong preferential adsorption to the
substrate used. The thiol molecules adsorb readily from solution
onto the gold, creating a dense monolayer with the tail group
pointing outwards from the surface. By using thiol molecules with
different tail groups, the resulting chemical surface functionality
can be varied within wide limits. It may be possible to chemically
modify the tail groups by performing reactions after assembly of
the SAM. For example, a simple incubation reaction of the thiol or
silane activated gold wire with the appropriate antibody, enzyme,
or DNA molecule can take place in order to achieve specific
detection.
Another option for the production of a biosensor within the hollow
fibre is to coat the entire inner surface of the hollow fibre with
gold using vapour deposition. The thickness of deposition can be
controlled to a depth of approximately 10 nm; this means the
coating does not interfere with the filtration function of the
hollow fibre. There are two methods for vapour deposition--chemical
and physical. Upon completion of chemical deposition the gold is
then coated with silanes or SAMs and a biomolecule and connected
via contact pads (80) to the data handling system as described
previously. Again different coatings on different fibres within the
manifold allow for the detection of multiple analytes. The benefits
to this methodology include no need to manipulate electrode wires
through the membrane, increase the number of active sites for
coating of specific biomolecules, increased sensitivity and
increased surface area.
Recent developments in polymer chemistry now mean that it is no
longer necessary to be restricted to metal based wires for
conduction and electrochemistry. A range of recently developed long
chain polymers (e.g. triisopropylsilyl) have been found to have
considerable conductivities. The availability of chemical groups on
the surface of the polymer means that proteins can be immobilised
directly (e.g. in chemical reactions) onto the polymer wire without
surface modification.
The hollow fibre membranes may be secured into a polymer casing
using standard techniques as described in WO 01/11006. Following
activation of the gold or polymer electrode (20) with a first
member of a specific binding pair (antibodies raised against E.
coli 0157 H7 or oligonucleotides specific for Salmonella
typhimurium) the electrodes (20, 40) are fed into the lumen (50) of
the hollow fibre membrane (60). The electrodes (20, 40) are then
secured with silver conducting paint to the contact pads (80). For
the initial filtration of the sample, the contact pads (80)
connected to the pre-amp (180) are sealed such that the sample
(220) is forced through the membrane (60) walls. During the
filtration process the specific analyte interacts with the bound
biosensor molecule (230) and generates a signal. However, the
ability of the total analyte population to be detected on a single
pass is limited and dependent on the specificity of the
analyte/biosensor interaction. These limitations may be due to flow
rate of the liquid across the bio-sensor or the surface area of the
bio-sensor or the affinity of the analyte for the bio-sensor. The
present invention allows for the repeat detection of analyte that
failed to be detected on first passage. Analyte which is not
detected is entrapped within the lumen of the hollow fibre membrane
(60) due to the pore size restriction properties of the membrane.
For example E. coli not bound to the anti-E. coli antibody coated
electrode will not pass through the membrane (60) walls but will
remain within the membrane (60) structure as a filtrand. The
entrapped bacteria are easily dislodged from the membrane walls by
a simple backflush mechanism, as described in WO 01/11006. To
ensure that there is no re-uptake of filtrate the system must
become open ended. The contact end cap (240) is replaced at this
stage with a contact pad (80) that is not sealed i.e. liquid can
pass through the length of the membrane (60) without being forced
through the membrane walls. The backflush liquid (250) used can be
a biological buffer such as PBS (phosphate buffered saline) or
Tris-BDTA. The buffer is applied into the membrane via the sample
entry port (260) using a syringe (not shown). Upon resuspension of
the filtrand the micro-organism can be detected by the detector.
This method facilitates the detection of a single micro-organism
within a liquid stream, which can be applied to liquids such as
beer, water, fruit juices in such areas as Clean In Place (CIP)
rinse water testing, final product sterility and process
control.
In a third embodiment of the present invention, multiple array
detector systems are used to detect multiple micro-organisms at the
same time. Within the food industry there are a number of
micro-organisms that are screened for regularly within wash water
and process lines, for example Escherichia coli, Salmonella
typhimurium and Listeria monocytogensis. By coating separate gold
or polymer electrodes with antibodies or oligonucleotides specific
for the micro-organism a multiple detector can be prepared. A
single wire for each of the specified microbes is fed into the
hollow fibre membrane and the detector constructed as described
previously. In order to determine the specific analyte being
detected, different contact pads for each of the specific analytes
is typically required. A computer is able to differentiate the data
from each of the contact pads, such that if E. coli is present, a
conductivity change is detected from the contact pad connected to
this specific electrode. The presence or absence of another
micro-organism is detected via the relevant contact pad.
In a fourth embodiment of the present invention, multiple
micro-organisms may be detected using multiple electrodes combined
with the use of secondary biosensor systems i.e. enzyme assay
systems. The electrodes may be coated using SAMs with a specific
antibody/DNA for the relevant microbe whereby a single electrode
can be coated with either one specific antibody or DNA, or multiple
antibodies or DNA. The coating of an electrode with SAMs is
detailed below. Self assembled monolayers (SAMs) comprising various
functionalised alkane thiols do not bind to one another. Their
chemical interaction is with the gold surface. To bind various
antibodies along a length of the electrode requires the use of
different functional groups on the outer region of the SAM such as
amine groups or carboxylic acid groups that will interact with the
antibodies. For example if three antibody types were to be placed
onto the electrode, each region of the electrode would be coated
with the appropriate SAM (one region at a time). To prevent SAMs
from binding out with the target region a priming agent or blocking
agent (eg photoresist) would be used for specific and defined
areas. The electrode would be immersed into the SAM solution for a
defined time period (dependent upon the kinetics of the SAM). The
next target region can then be coated in a similar manner. There
will be no binding within the previous region from the second SAM
solution. This can be repeated for the appropriate number of
antibody regions. The antibodies can be bound to the SAM on the
electrode by immersion within the specific antibody solution or a
mixed antibody solution. The SAM with specific functional group for
each antibody will only bind to its appropriate ligand.
Detection of antigen binding to an antibody, or nucleic acid
hybridisation will be detected using the following methods:
Secondary antibody conjugates or nucleic acid-enzyme conjugates may
be employed, wherein the electrode may be washed after sampling has
been conducted. For example, detection of E. coli may be through
the use of an anti-E. coli-luciferase secondary antibody conjugate
which may be passed through the filter device, which may then bind
to the bacteria immobilised on the electrode. When the substrate
luciferin is passed through the filter device, the reaction may be
detected by monitoring CO.sub.2 generation, for example. In the
same way, Salmonella typhimurium may be detected by using an
anti-S. typhimurium alkaline phosphatase secondary antibody
conjugate, such that phosphate release could be measured in the
presence of an appropriate substrate. There are numerous
enzyme/biosensor systems that can be adapted to this purpose.
Another method that can be used to detect antigen binding to an
antibody, or nucleic acid hybridisation is direct measurement using
specific redox potential of each antibody/antigen binding (or
nucleic acid hybrids) as sampling is occurring ie real time
analysis. This will require the specific redox potential to be
determined for each complex prior to analysis such that a shift in
potential can be directly related to a specific antigen binding or
a specific nucleic acid hybridisation event.
Another method that can be used to detect antigen binding to an
antibody, or nucleic acid hybridisation could be by using reference
electrodes for each antibody/antigen complex or nucleic acid hybrid
in order to obtain real time control analysis i.e. for each
antibody or nucleic acid used there would be a reference electrode
that may be fed into the detection unit which would give a real
time reference output of the expected redox potential if the sample
contains the specific antigen or nucleic acid target.
In a fifth embodiment of the present invention, multiple airborne
bio/chemical warfare agents are detected in a single filtration
unit, using multiple electrodes coated with different affinity
ligands, for example using electrodes coated with antibodies raised
against Bacillus anthracis (the agent causing anthrax), antibodies
raised against Ebola virus, and acetylcholinesterase enzyme (for
detection of chemical nerve agents such as VX and sarin). The
collected air sample may be placed into a suitable buffer such as a
mild organic solvent, saline solution or surfactant. The sarin/VX
may dissolve within the appropriate buffer. The buffer may then be
passed through the membrane unit where the bio/chemical warfare
agents bind to their appropriate affinity ligand on the
specifically coated electrodes. The detection would be as
above.
Detectors can be prepared to recognise alterations in the levels of
bio-luminescence, electrochemical activity, fluorescence using
fluorophores, chemi-luminescence, radiochemicals such as alpha,
beta and gamma particles, antibodies-antigen interaction,
protein/enzyme interaction, nucleic acid hybridisation.
* * * * *